DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications.

Phage display links the phenotype of displayed polypeptides with the DNA sequence in the phage genome and offers a universal method for the discovery of proteins with novel properties. However, the display of large multisubunit proteins on phages remains a challenge. A majority of protein display systems are based on monovalent phagemid constructs, but methods for the robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼ 200 kDa tetrameric l-asparaginase protein on M13 and fd phages produced by ligation of SpyCatcher-Asparaginase fusion (ScA) and PEGylated-ScA (PEG-ScA) to barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 or p8 coat proteins yielded constructs with five copies of ScA displayed on p3 (ScA-p3), ∼100 copies of ScA on p8 protein (ScA-p8) and ∼300 copies of PEG-ScA on p8 protein (PEG-ScA-p8). Display constructs of different valencies and chemical modifications on protein (e.g., PEGylation) can be injected into mice and analyzed by deep sequencing of the DNA barcodes associated with phage clones. In these multiplexed studies, we observed a density and protein-dependent clearance rate in vivo. Our observations link the absence of PEGylation and increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of l-asparaginase on phages could be used to study the circulation life of this protein in vivo, and such an approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multisubunit proteins in vivo.

Inexpensive anti-cysticercosis vaccine: S3Pvac expressed in heat inactivated M13 filamentous phage proves effective against naturally acquired Taenia solium porcine cysticercosis.

In search of reducing vaccine production costs', a recombinant M13 phage version of the anti-cysticercosis tripeptide vaccine (S3Pvac) was developed. The efficacy of S3Pvac-Phage vs. placebo was evaluated in a randomized trial that included 1,047 rural pigs in 16 villages of Central Mexico. Three to five months after vaccination 530 pigs were examined by tongue inspection. At 5-27 months of age, 331 pigs (197 vaccinated/134 controls) were inspected at necropsy. Vaccination reduced 70% the frequency of tongue cysticercosis and, based on necropsy, 54% of muscle-cysticercosis and by 87% the number of cysticerci.

Monoclonal antibodies against the major coat protein of filamentous phage as a useful analytical tool for bacteriophage peptide/protein display.

Four mouse monoclonal antibodies (MAbs) that react with filamentous M13KO7 and R408 phage were obtained. Three of these MAbs (two IgG2a and one IgG3) recognize linear sequences of the p8 main structural coat protein, and one (IgG2a) identifies a putatively conformational epitope, as suggested by Western blot. These MAbs also react with recombinant phage expressing peptide antigens fused to p8, and are though useful reagents for peptide/protein phage display screening based methods. The latter was shown in an enzyme-linked immunoadsorbent assay (ELISA) and a visual immunoassay where one of the anti-p8 MAbs was used to capture recombinant phages displaying a peptide characteristic of the Hepatitis B virus surface antigen or a Dengue virus-related peptide antigen.